Methods and compositions for the generation and use of conformation-specific antibodies

ABSTRACT

The present invention features methods and compositions for the generation and use of conformation-specific antibodies or fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/325,013, filed Jul. 7, 2014, which is a divisional of U.S. patentapplication Ser. No. 13/504,700, filed Oct. 11, 2012, which is theNational Stage of International Application No. PCT/US2010/054077, filedOct. 26, 2010, which claims the benefit of U.S. Provisional PatentApplication No. 61/255,341, filed Oct. 27, 2009.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers NIHGM058556, AG0178870, and AG022082. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

In general, the invention relates to methods and compositions for thegeneration and use of conformation-specific antibodies or fragmentsthereof.

Protein phosphorylation is a key cellular signaling mechanism thatinduces changes in protein conformation. For example, thephosphorylation of specific serine or threonine residues thatimmediately precede a proline residue (Ser/Thr-Pro motif) is a centralregulatory mechanism in the cell. The unique stereochemistry of theproline residue means that the peptidyl-prolyl bond of the Ser/Thr-Promotif can adopt two different conformational states (i.e., a cisconformation or a trans conformation). Peptidyl-prolyl cis/transisomerases (PPIases) specifically catalyze the cis/trans isomerizationof Ser/Thr-Pro motifs and, thus, regulate the structure of theseproteins between the two distinct conformations.

Pin1 is a PPIase that specifically catalyzes the cis/trans isomerizationof certain phosphorylated Ser/Thr-Pro (pSer/Thr-Pro) motifs. Theidentification of Pin1 as a phosphorylation-specific PPIase led to theunderstanding of a new signaling mechanism, whereby Pin1 catalyticallyregulates the conformation of its substrates after their phosphorylationto further control protein function. Indeed, Pin1-catalyzedconformational changes control many protein functions. Moreover, Pin1 istightly regulated by multiple mechanisms, and the deregulation of Pin1plays a pivotal role in some human diseases (e.g., cancer, Alzheimer'sdisease, and asthma). Given the completely different conformation of cisand trans Ser/Thr-Pro motifs (e.g., phosphorylated and nonphosphorylatedSer/Thr-Pro motifs), the generation of conformation-specific antibodieswould allow for the diagnosis and treatment of disorders associated withspecific protein conformations.

Thus, there exists a need in the art for conformation-specificantibodies that specifically bind to a cis or trans conformation of aXaa-Pro (e.g., Ser/Thr-Pro or phosphorylated Ser/Thr-Pro) motif of apolypeptide, where Xaa may be any amino acid residue.

SUMMARY OF THE INVENTION

In general, the present invention features methods and compositions forthe generation and use of conformation-specific antibodies or fragmentsthereof.

In a first aspect, the invention features an isolatedconformation-specific antibody or fragment thereof that specificallybinds to a Xaa-Pro motif of a polypeptide, wherein the peptidyl-prolylbond of the Xaa-Pro motif is in a cis conformation or a transconformation and wherein Xaa is any amino acid residue. In oneembodiment, the antibody is a monoclonal antibody or a polyclonalantibody.

In a second aspect, the invention features an isolatedconformation-specific antibody or fragment thereof that specificallybinds to a Xaa-Pro motif of a polypeptide, wherein the peptidyl-prolylbond of the Xaa-Pro motif is in a cis or trans conformation and Xaa isany amino acid residue, produced by a process that includes: (i)providing an antibody library; (ii) contacting the antibody library witha polypeptide that has a Xaa-Pro motif; (iii) determining binding of anantibody from the antibody library to the Xaa-Pro motif of thepolypeptide, wherein the antibody specifically binds to the cis or transconformation of the Xaa-Pro motif of the polypeptide; and (iv) isolatingthe antibody, wherein the antibody is a conformation-specific antibody.In one embodiment, the antibody library is a synthetic antibody library.

In a third aspect, the invention features a method of generating aconformation-specific antibody or fragment thereof that specificallybinds to a Xaa-Pro motif of a polypeptide, where Xaa is any amino acidresidue. The method includes: (i) administering aproline-analog-containing antigenic peptide to a host animal, whereinthe antigenic peptide has a Xaa-Pro motif; (ii) isolating antiseracontaining the antibody or fragment thereof produced in the animal; and(iii) purifying the conformation-specific antibody or fragment thereoffrom the antisera.

In one embodiment of the first, second, or third aspect, the antibody orfragment thereof binds to the cis conformation of the Xaa-Pro motif ofthe polypeptide with at least 10- to 100-fold greater affinity than tothe trans conformation of the Xaa-Pro motif of the polypeptide. In analternate embodiment, the antibody or fragment thereof binds to thetrans conformation of the Xaa-Pro motif of the polypeptide with at least10- to 100-fold greater affinity than to the cis conformation of theXaa-Pro motif of the polypeptide. In another embodiment of the first,second, or third aspect, the polypeptide is a PPIase substrate. ThePPIase substrate may be a Pin1 substrate (e.g., NIMA, RAB4, CDC25, WEE1,PLK1, MYT1, CDC27, CENP-F, Incenp, RBP1, NHERF-1, KRMP1, CK2, TopoIIα,DAB2, p54nrb, SiI, EMI1, cyclin D1, Ki67, c-Myc, cyclin E, c-Jun,β-catenin, Cf-2, NF-κB, RAF1, c-Fos, RARα, AIB1/SRC-3, HBx, STAT3, p53,Bcl-2, p73, BimEL, p66^(Shc), CHE1, tau, amyloid precursor protein(APP), APP fragment, synphilin-1, gephyrin, MCL1, NFAT, AUF1, IRF3, BTK,SIN3-RPD3, or hSpt5).

In a fourth aspect, the invention features a method of purifying aconformation-specific antibody or fragment thereof that specificallybinds to a Xaa-Pro motif of a polypeptide, wherein the peptidyl-prolylbond of the Xaa-Pro motif is in a cis conformation and wherein Xaa isany amino acid residue. The method includes: (i) adsorbing aproline-analog-containing antigenic peptide to a support (e.g., achromatographic support), wherein the antigenic peptide has a Xaa-Promotif and wherein the peptidyl-prolyl bond of the Xaa-Pro motif of theantigenic peptide is in a cis conformation; (ii) contacting theantigenic peptide on the support with antisera containing the antibodyor fragment thereof produced in a host animal, wherein the contacting isunder conditions allowing the conformation-specific antibody or fragmentthereof to specifically bind to the antigenic peptide; and (iii) elutingthe conformation-specific antibody from the antigenic peptide adsorbedto the support.

In one embodiment of the third or fourth aspect, the proline analog ishomoproline, pipecolic acid (PIP), dimethyl proline (DMP),azetidine-2-carboxylic acid (Aze), tert-butyl-L-proline (TBP),trans-4-fluoro-L-proline (t-4F-Pro), or cis-4-fluoro-L-proline(c-4F-Pro). In another embodiment, the peptidyl-prolyl bond of theXaa-Pro motif of the antigenic peptide is fixed in the cis conformationor the trans conformation. In a further embodiment, the antigenicpeptide is at least 8 amino acid residues in length (e.g., between 8 and20 amino acid residues in length). In another embodiment, theconformation-specific antibody is a monoclonal antibody or a polyclonalantibody. In a further embodiment, the host animal is a rabbit.

In a fifth aspect, the invention features a method of diagnosing asubject with a disorder, wherein the disorder is associated with aderegulation of PPIase activity. The method includes: (i) contacting asample from the subject with a conformation-specific antibody orfragment thereof that specifically binds to a Xaa-Pro motif of apolypeptide present in the sample, wherein the peptidyl-prolyl bond ofthe Xaa-Pro motif is in a cis conformation and wherein Xaa is any aminoacid residue; (ii) quantitating the amount of conformation-specificantibody or fragment thereof bound to the Xaa-Pro motif in the cisconformation; and (iii) comparing the amount of Xaa-Pro motif in the cisconformation in the sample to the amount of Xaa-Pro motif in the cisconformation found in subjects diagnosed with the disorder or subjectsnot diagnosed with the disorder, wherein an increase in the amount ofXaa-Pro motif in the cis conformation in the subject, in comparison tothe amount of Xaa-Pro motif in the cis conformation in subjects notdiagnosed with the disorder, indicates that the subject may be diagnosedwith the disorder.

In a sixth aspect, the invention features a method of diagnosing asubject with a disorder, wherein the disorder is associated with aderegulation of PPIase activity. The method includes: (i) contacting asample from the subject with a conformation-specific antibody orfragment thereof that specifically binds to a Xaa-Pro motif of apolypeptide present in the sample, wherein the peptidyl-prolyl bond ofthe Xaa-Pro motif is in a trans conformation and wherein Xaa is anyamino acid residue; (ii) quantitating the amount ofconformation-specific antibody or fragment thereof bound to the Xaa-Promotif in the trans conformation; and (iii) comparing the amount ofXaa-Pro motif in the trans conformation in the sample to the amount ofXaa-Pro motif in the trans conformation found in subjects diagnosed withthe disorder or subjects not diagnosed with the disorder, wherein anincrease in the amount of Xaa-Pro motif in the trans conformation in thesubject, in comparison to the amount of Xaa-Pro motif in the transconformation in subjects not diagnosed with the disorder, indicates thatthe subject may be diagnosed with the disorder.

In a seventh aspect, the invention features a method of treating asubject with a disorder, wherein the disorder is associated with aderegulation of PPIase activity. The method includes administering tothe subject a pharmaceutical composition that includes aconformation-specific antibody or fragment thereof that specificallybinds to a Xaa-Pro motif of a polypeptide present in the subject,wherein Xaa is any amino acid residue and wherein theconformation-specific antibody is present in amount sufficient to treatthe disorder. In another embodiment, the method may further includeadministering an additional therapeutic agent (e.g., a chemotherapeuticagent).

In one embodiment of the fifth, sixth, or seventh aspect, the PPIase isPin1. In another embodiment, the disorder is a cell proliferationdisorder (e.g., cancer), a neurological disorder (e.g., Alzheimer'sdisease), asthma, a microbial infection, or aging or an aging-relateddisorder.

In an eighth aspect, the invention features an antigenic peptide with aXaa-Pro motif that specifically binds to an antibody or fragmentthereof, wherein Xaa is any amino acid residue and wherein the antibodyor fragment thereof binds to the cis conformation of the Xaa-Pro motifof the antigenic peptide with at least 10- to 100-fold greater affinitythan to the trans conformation of the Xaa-Pro motif of the polypeptide.

In a final aspect, the invention features an antigenic peptide with aXaa-Pro motif that specifically binds to an antibody or fragmentthereof, wherein Xaa is any amino acid residue and wherein the antibodyor fragment thereof binds to the trans conformation of the Xaa-Pro motifof the antigenic peptide with at least 10- to 100-fold greater affinitythan to the cis conformation of the Xaa-Pro motif of the polypeptide.

In one embodiment of the eight or final aspect, the proline of theXaa-Pro motif is a proline analog (e.g., homoproline, pipecolic acid(PIP), dimethyl proline (DMP), azetidine-2-carboxylic acid (Aze),tert-butyl-L-proline (TBP), trans-4-fluoro-L-proline (t-4F-Pro), orcis-4-fluoro-L-proline (c-4F-Pro)). In another embodiment, thepeptidyl-prolyl bond of the Xaa-Pro motif of the antigenic peptide isfixed in the cis conformation or the trans conformation. In a furtherembodiment, the antigenic peptide is at least 8 amino acid residues inlength (e.g., between 8 and 20 amino acid residues in length). In oneembodiment, the antigenic peptide includes a Xaa-Pro motif of a Pin1substrate (e.g., a Pin1 substrate listed in Table 1). In an alternateembodiment, the antigenic peptide includes a Xaa-Pro motif of apolypeptide selected from steroid receptors, c-Myb, H3P30, H3P38, Itk,5-hydroxytryptamine type 3 (5-HT3) receptors, the phage tip protein G3P,the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1)virion, intracellular calcium release channel, CrkII/CrkL proteins,centrosome protein 55 kDa (Cep55), the retroviral Re1 proteins, PKB/Akt,human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein, Stat3,HER2/Neu, Notch, FAK, FOXO, PML, C/EBP, and SMRT.

In one embodiment of any of the above aspects, Xaa is a serine orthreonine amino acid residue. In another embodiment, Xaa isphosphorylated. In an alternative embodiment, Xaa is not phosphorylated.

As used herein, the term “abnormal cell growth” is intended to includecell growth that is undesirable or inappropriate. Abnormal cell growthalso includes proliferation that is undesirable or inappropriate (e.g.,unregulated cell proliferation or undesirably rapid cell proliferation).Abnormal cell growth can be benign and result in benign masses of tissueor cells (e.g., benign tumors). Many art-recognized conditions areassociated with such benign masses or benign tumors, including, forexample, diabetic retinopathy, retrolental fibrioplasia, neovascularglaucoma, psoriasis, angiofibromas, rheumatoid arthritis, hemangiomas,and Karposi's sarcoma. Abnormal cell growth can also be malignant andresult in malignancies, malignant masses of tissue or cells, ormalignant tumors. Many art-recognized conditions and disorders areassociated with malignancies, malignant masses, and malignant tumors,including, for example, cancer and carcinoma.

By “antibody” is meant monoclonal antibodies, polyclonal antibodies,humanized antibodies, chimeric antibodies, recombinant antibodies,multispecific antibodies, and antibody fragments. The antibody may be,for example, a conformation-specific antibody (e.g., an antibody thatbinds to the cis or trans conformation of a Xaa-Pro motif). An antibodyspecifically binds to an antigen. The antibody may also be anon-immunoglobulin binding polypeptide.

By “antigen” is meant a molecule to which an antibody can selectivelybind. The target antigen may be a protein (e.g., an antigenic peptide),carbohydrate, nucleic acid, lipid, hapten, or other naturally occurringor synthetic compound. The target antigen may be a polypeptide (e.g., apolypeptide containing a Xaa-Pro motif (e.g., a phosphorylated ornonphosphorylated Ser/Thr-Pro motif)) or peptide mimics (e.g., apolypeptide containing a Xaa-homoproline motif (e.g., a phosphorylatedor nonphosphorylated Ser/Thr-homoproline motif)). An antigen may also beadministered to an animal to generate an immune response in the animal.

By “binding affinity” is meant the strength of the total noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen or antigenicpeptide). Unless otherwise indicated, as used herein, “binding affinity”refers to intrinsic binding affinity, which reflects a specificinteraction between members of a binding pair (e.g., antibody andantigen). The affinity of a molecule X for its partner Y can generallybe represented by the dissociation constant (K_(d)). Affinity can bemeasured by standard methods known in the art, including those describedherein. A low-affinity complex contains an antibody that generally tendsto dissociate readily from the antigen, whereas a high-affinity complexcontains an antibody that generally tends to remain bound to the antigenfor a longer duration.

By “biological sample” or “sample” is meant solid and fluid samples.Biological samples may include cells, protein or membrane extracts ofcells, blood or biological fluids including, e.g., ascites fluid orbrain fluid (e.g., cerebrospinal fluid (CSF)). Examples of solidbiological samples include samples taken from feces, the rectum, centralnervous system, bone, breast tissue, renal tissue, the uterine cervix,the endometrium, the head or neck, the gallbladder, parotid tissue, theprostate, the brain, the pituitary gland, kidney tissue, muscle, theesophagus, the stomach, the small intestine, the colon, the liver, thespleen, the pancreas, thyroid tissue, heart tissue, lung tissue, thebladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue,adrenal tissue, testis tissue, the tonsils, and the thymus. Examples ofbiological fluid samples include samples taken from the blood, serum,CSF, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus,bone marrow, lymph, and tears. Samples may be obtained by standardmethods including, e.g., venous puncture and surgical biopsy. In certainembodiments, the biological sample is a breast, lung, colon, or prostatetissue sample obtained by needle biopsy.

By “cancer” and “cancerous” is meant the physiological condition inmammals that is typically characterized by abnormal cell growth.Included in this definition are benign and malignant cancers, as well asdormant tumors or micro-metastases. Examples of cancer include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.More particular examples of such cancers include, e.g., prostate cancer,squamous cell cancer, small-cell lung cancer, non-small-cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma, gastric cancer, melanoma, and various typesof head and neck cancer.

By “cell proliferation disorder” is meant a disorder associated withabnormal cell growth. Exemplary cell proliferative disorders includecancer (e.g., benign and malignant), benign prostatic hyperplasia,psoriasis, abnormal keratinization, lymphoproliferative disorders,rheumatoid arthritis, arteriosclerosis, restenosis, diabeticretinopathy, retrolental fibrioplasia, neovascular glaucoma,angiofibromas, hemangiomas, Karposi's sarcoma, and neurodegenerativedisorders. Cellular proliferative disorders are described, for example,in U.S. Pat. Nos. 5,639,600, 7,087,648, and 7,217,737, herebyincorporated by reference.

By “conformation-specific antibody” is an antibody or fragment thereofthat recognizes and specifically binds to a particular conformation(e.g., a conformational isomer or conformer) of its complementaryantigen. For example, as described herein, the conformation-specificantibody may specifically bind to the cis conformation of a Xaa-Promotif, but will not specifically bind to the trans conformation of theXaa-Pro motif, where Xaa is any amino acid residue (e.g., serine orthreonine). In this case, the conformation-specific antibody will have,for example, at least 10- to 100-fold greater affinity to the cisconformation than to the trans conformation of a Xaa-Pro motif.Conversely, the conformation-specific antibody may specifically bind tothe trans conformation of a Xaa-Pro motif, but will not specificallybind to the cis conformation of the Xaa-Pro motif, where Xaa is anyamino acid residue (e.g., serine or threonine). In certain embodiments,the Ser/Thr-Pro motif may be phosphorylated (i.e., pSer/Thr-Pro).

By “disorder” is meant any condition that may be treated, inhibited,diagnosed, or screened for according to the methods of the inventiondescribed herein. By “disorder associated with a deregulation of PPIaseactivity” is meant a disorder in which PPIase (e.g., Pin1) activity ismodulated (e.g., upregulated or downregulated). Non-limiting examples ofdisorders associated with a deregulation of PPIase activity to betreated, inhibited, diagnosed, or screened for by the methods andcompositions described herein include, e.g., cellular proliferationdisorders (e.g., cancer), neurological disorders (e.g., Alzheimer'sdisease), aging-related disorders, asthma, and microbial infections.

By “fragment” is meant a portion of a nucleic acid or polypeptide (e.g.,an antibody) that contains at least, e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or more of the entire length of the nucleic acid orpolypeptide. A nucleic acid fragment may contain, e.g., 10, 20, 30, 40,50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 4000, 4500, or5000 nucleotides or more nucleotides, up to the full length of thenucleic acid. A polypeptide fragment may contain, e.g., 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 aminoacids or more amino acids, up to the full length of the polypeptide.Fragments useful in the therapeutic methods of the invention include,e.g., fragments of conformation-specific antibodies that retainbiological activity (e.g., fragments that bind to a specificconformational state). Fragments can be modified as described herein andas known in the art.

By “humanized antibody” is meant an immunoglobulin amino acid sequencevariant or fragment thereof that is capable of binding to apredetermined antigen. The antibody may contain both the light chain, aswell as at least the variable domain of a heavy chain. The antibody alsomay include the CH1, hinge, CH2, CH3, or CH4 regions of the heavy chain.The humanized antibody comprises a framework region (FR) havingsubstantially the amino acid sequence of a human immunoglobulin and acomplementarity determining region (CDR) having substantially the aminoacid sequence of a non-human immunoglobulin.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. In general, thehumanized antibody may comprise substantially all of at least one, andtypically two, variable domains (e.g., Fab, Fab′, F(ab′)₂, Fabc, or Fv)in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody may comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. By“complementarity determining region (CDR)” is meant the threehypervariable sequences in the variable regions within each of theimmunoglobulin light and heavy chains. By “framework region” is meantthe sequences of amino acids located on either side of the threehypervariable sequences of the immunoglobulin light and heavy chains.

The term “monoclonal antibody,” as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies(i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts). Monoclonal antibodies are highly specific, beingdirected against a single antigenic site. Furthermore, in contrast toconventional (e.g., polyclonal) antibody preparations, which typicallyinclude different antibodies directed against different determinants(e.g., epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al. (see, e.g., Nature 256: 495, 1975) or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al. (Nature 352: 624-628, 1991) andMarks et al. (J. Mol. Biol. 222: 581-597, 1991), for example.

By “neurological disorder” is meant a disturbance in the structure orfunction of the nervous system resulting from a developmentalabnormality, disorder, injury, or toxin. Exemplary neurologicaldisorders include Alzheimer's disease (AD), mild cognitive impairment(MCI), Parkinson's disease (PD), multiple sclerosis (MS), musculardystrophy, corticobasal degeneration, dementia pugilistica, Down'ssyndrome, frontotemporal dementias, myotonic dystrophy, Niemann-Pickdisease, Pick's disease, prion disease, progressive supranuclear palsy,subacute sclerosing panencephalistis, convulsive disorders (e.g.,epilepsy), vascular dementia, age-related dementia, head trauma, stroke,neurofibromatosis, Lewy body disease, amyotrophic lateral sclerosis(ALS), peripheral neuropathies, and macular degeneration.

By “pharmaceutically acceptable carrier” is meant a carrier that isphysiologically acceptable to the treated subject while retaining thetherapeutic properties of the composition (e.g., theconformation-specific antibody) with which it is administered. Oneexemplary pharmaceutically acceptable carrier substance is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and are described, for example, inRemington's Pharmaceutical Sciences (20^(th) edition, ed. A. Gennaro,2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

By “protein,” “polypeptide,” “polypeptide fragment,” or “peptide” ismeant any chain of more than two amino acid residues, regardless ofpost-translational modification (e.g., glycosylation orphosphorylation), constituting all or part of a naturally occurringpolypeptide or peptide or constituting a non-naturally occurringpolypeptide or peptide. A polypeptide or peptide may be said to be“isolated” or “substantially pure” when physical, mechanical, orchemical methods have been employed to remove the polypeptide fromcellular constituents. An “isolated polypeptide” (e.g., an isolatedantibody), “substantially pure polypeptide,” or “substantially pure andisolated polypeptide” is typically considered removed from cellularconstituents and substantially pure when it is at least 60% by weightfree from the proteins and naturally occurring organic molecules withwhich it is naturally associated. The polypeptide may be at least 75%,80%, 85%, 90%, 95%, or 99% by weight pure. A substantially purepolypeptide (e.g., a substantially pure antibody or fragment thereof)may be obtained by standard techniques, for example, by extraction froma natural source (e.g., cell lines or biological fluids), by expressionof a recombinant nucleic acid encoding the polypeptide, or by chemicallysynthesizing the polypeptide. Purity can be measured by any appropriatemethod, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. Alternatively, a polypeptide isconsidered isolated if it has been altered by human intervention, placedin a location that is not its natural site, or if it is introduced intoone or more cells.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound that hassubstantially the same structural and/or functional characteristics ofthe peptides (e.g., antigenic peptides) or polypeptides of theinvention. The mimetic can be either entirely composed of synthetic,non-natural analogs of amino acids or may be a chimeric molecule ofnatural amino acids and non-natural analogs of amino acids. The mimeticcan also incorporate any amount of conservative substitutions, as longas such substitutions do not substantially alter the mimetic's structureor activity.

By “proline analog” is meant a molecule substantially similar infunction to either an entire proline amino acid residue or to a fragmentthereof. For example, the present invention contemplates the use ofproline analogs wherein a side chain is lengthened or shortened whilestill providing a carboxyl, amino, or other reactive precursorfunctional group, as well as proline analogs having variant side chainswith appropriate functional groups. Exemplary proline analogs include,without limitation, homoproline (i.e., pipecolic acid (PIP)), dimethylproline (DMP), azetidine-2-carboxylic acid (Aze), tert-butyl-L-proline(TBP), trans-4-fluoro-L-proline (t-4F-Pro), or cis-4-fluoro-L-proline(c-4F-Pro).

By “reduce or inhibit” is meant the ability to cause an overall decreaseof 20% or greater, of 50% or greater, or of 75%, 80%, 85%, 90%, 95%, orgreater. For therapeutic applications, to “reduce or inhibit” can referto the symptoms of the disorder being treated or the presence or extentof a disorder being treated. For diagnostic or monitoring applications,to “reduce or inhibit” can refer to a decrease in the level of proteinor nucleic acid detected by the diagnostic or monitoring assays.

By “reference” is meant any sample, standard, or level that is used forcomparison purposes. A “normal reference sample” can be a prior sampletaken from the same subject prior to the onset of a disorder (e.g., acellular proliferation disorder or a neurological disorder), a samplefrom a subject not having the disorder, a subject that has beensuccessfully treated for the disorder, or a sample of a purifiedreference polypeptide at a known normal concentration. By “referencestandard or level” is meant a value or number derived from a referencesample. A normal reference standard or level can be a value or numberderived from a normal subject that is matched to a sample of a subjectby at least one of the following criteria: age, weight, disease stage,and overall health. In one example, a normal reference level of, forexample, a polypeptide indicative of a disorder or a conformation of apolypeptide indicative of a disorder, is less than 5 ng/ml in a serumsample, less than 4 ng/ml, less than 3 ng/ml, less than 2 ng/ml, or lessthan 1 ng/ml in a serum sample. A “positive reference” sample, standard,or value is a sample, standard, value, or number derived from a subjectthat is known to have a disorder (e.g., a cellular proliferationdisorder or a neurological disorder) that is matched to a sample of asubject by at least one of the following criteria: age, weight, diseasestage, and overall health. For example, a positive reference value for,e.g., a polypeptide indicative of a disorder, is greater than 5 ng/mlserum, greater than 10 ng/ml serum, greater than 20 ng/ml, greater than30 ng/ml, greater than 40 ng/ml, or greater than 50 ng/ml serum.

By “specifically binds” is meant a molecule (e.g., an antibody) whichrecognizes and binds another molecule (e.g., an antigen), but that doesnot substantially recognize and bind other molecules. In one example, anantibody that specifically binds the cis conformation of a Xaa-Pro motifof a polypeptide does not specifically bind the trans conformation of aXaa-Pro motif of a polypeptide, where Xaa is any amino acid residue(e.g., serine or threonine). The term “specific binding,” “specificallybinds to,” or is “specific for” a particular molecule (e.g., apolypeptide, an epitope on of a polypeptide, or a conformation of apolypeptide), as used herein, can be exhibited, for example, by amolecule having a Ka for the molecule to which it binds of at leastabout 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M,10⁻¹² M, or greater.

The term “specifically binds” may also refer to binding where a molecule(e.g., an antibody) binds to a particular polypeptide (e.g., apolypeptide containing a Xaa-Pro motif, where Xaa is any amino acidresidue (e.g., serine or threonine)), an epitope on a particularpolypeptide, or a conformation of a particular polypeptide (e.g., a cisconformation of a Xaa-Pro motif) without substantially binding to anyother polypeptide, polypeptide epitope, or polypeptide conformation(e.g., the trans conformation of a Xaa-Pro motif). For example, theconformation-specific antibody may have, for example, at least 10- to100-fold greater affinity (e.g., 10¹-, 10²-, 10³-, 10⁴-, 10⁵-, 10⁶-,10⁷-, 10⁸-, 10⁹-, or 10¹⁰-fold greater affinity) to one conformation(e.g., the cis conformation) than to another conformation (e.g., thetrans conformation) of, for example, a Ser/Thr-Pro motif.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “therapeutic amount” is meant an amount that, when administered to asubject suffering from a disorder (e.g., a cellular proliferativedisorder, a neurological disorder, asthma, or a microbial infection), issufficient to cause a qualitative or quantitative reduction in thesymptoms associated with the disorder.

By “treating” is meant administering a pharmaceutical composition fortherapeutic purposes or administering treatment to a subject alreadysuffering from a disorder to improve the subject's condition. By“treating a disorder” is meant that the disorder and the symptomsassociated with the disorder are, e.g., alleviated, reduced, cured, orplaced in a state of remission.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the Pin1-catalyzed conformationalswitch between the cis and trans conformation of pSer/Thr-Pro motifs.

FIGS. 2A and 2B are schematic diagrams showing thatphosphorylation-dependent prolyl cis-trans isomerization acts as amolecular timer in amyloid precursor protein (APP) processing and Aβproduction in Alzheimer's disease (AD). Phosphorylation of APP on theThr668-Pro motif occurs during mitosis in the cell cycle and is alsoincreased in AD brains. Before phosphorylation, the Thr668-Pro motif inAPP is in a trans conformation in a helix cap structure. Although thepThr668-Pro motif of APP is likely phosphorylated in trans by upstreamkinases, it has a tendency to be in cis, with the overall content being˜10% due to both destabilization of the trans isomer by loss of hydrogenbonds resulting from a local unfolding of a helix cap and tostabilization of the cis isomer by hydrogen bonds involving thephosphate. Pin1 accelerates both k_(cis to trans) _(cat) andk_(trans to cis) _(cat) by several orders of magnitude over the typicaluncatalyzed isomerization rates for pThr-Pro peptides, resulting in adramatic reduction in the average lifetime of both the cis (˜0.05 s) andtrans (˜0.5 s) isomeric states to fractions of a second, with thecatalyzed cis to trans rate being 10-fold faster than the catalyzedtrans to cis rate. This favors more non-amyloidogenic APP processing,reducing Aβ production (FIG. 2A). In contrast, without proper Pin1function, the cis pThr668-Pro motif may not be isomerized to trans,which might favor more amyloidogenic APP processing and Aβ production(FIG. 2B). Therefore, in collaboration with other AD factors, Pin1deregulation can promote non-amyloidogenic APP processing and Aβproduction.

FIGS. 3A and 3B are graphs showing the cis/trans content of a pThr-Propeptide and pThr-Prx peptide, respectively. FIG. 3C is a schematicdiagram showing the methodology for producing conformation-specificantibodies. FIG. 3D is a bar graph showing that only cis-specificantibodies recognized pT231-dmP tau peptide, assayed by ELISA. Both cis-and trans-specific antibodies recognized pT231-Pro tau peptide. Neitherthe cis- or trans-specific antibodies recognized nonphosphorylated taupeptide or pT231-Ala tau peptide. FIG. 3E is a series of Western blotsthat show that both cis- and trans-specific antibodies recognizedpT231-Pro-containing tau protein, but not Ala231-Pro-containing tauprotein.

FIGS. 4A and 4B are micrographs showing that the cis-, but not trans-,pT231-tau accumulated during tauopathy development, but is effectivelyreversed by Pin1 overexpression in mouse models. FIG. 4C is a series ofWestern blots that show that Pin1 overexpression in tau mice decreasesthe cis content, but increases the trans content, reversing thecis/trans ratio. The semiquantitative results of the Western blots areshown in FIG. 4D.

FIGS. 5A-D are a series of micrographs showing that cis-, but nottrans-, pT231-tau is elevated in human brain samples with mild cognitiveimpairment (MCI) and further accumulates as AD progresses. FIGS. 5A and5B are micrographs showing human brain samples immunostained with cis-or trans-pT231-tau antibodies. FIGS. 5C and 5D are micrographs showinghuman brain samples immunostained with cis-pT231-tau antibodies and TG3monoclonal antibody. TG3 antibody recognizes pT231-tau in theconformation that is only detected in AD brains, but not in normal humanbrains or MCI brains.

FIGS. 6A-C are a series of bar graphs showing that the cis/trans ratioof pT231-tau in cerebrospinal fluid (CSF) was elevated in advanced ADpatients with small individual variations. The cis- and trans-pT231-tauin CSF of late AD patients and controls were assayed in triplicate byELISA using cis- (FIG. 6A) or trans- (FIG. 6B) specific antibodies. FIG.6C is a bar graph showing the cis/trans ratio (“nd”: not detectable;“na”: not applicable).

DETAILED DESCRIPTION

We describe the generation, purification, and use ofconformation-specific antibodies. In particular, we have generated andpurified antibodies specific to the cis or trans conformation of thephosphorylated Ser/Thr-Pro motif using antigenic formulations containingproline analogs. The same strategy can be used to generate and purifyantibodies specific to the cis or trans conformation of a Xaa-Pro motif,where Xaa is any amino acid residue. Such conformation-specificantibodies may be useful for the treatment, diagnosis, and monitoring ofcertain disorders, including, e.g., cancer, Alzheimer's disease, asthma,inflammation, immune diseases, and aging.

PPIases and the Cis/Trans Conformation of PPIase Substrates

Proline is an amino acid residue unique in its ability to adopt eitherthe cis or trans conformation. Due to the relatively large energybarrier of its isomerization (ε^(u)=14 to 24 kcal mol⁻¹), uncatalyzedisomerization is a slow process, but may be accelerated by PPIases (see,e.g., FIGS. 1 and 2). PPIases facilitate protein folding and include,for example, cyclophilins (Cyps), FK506-binding proteins (FKBPs), andparvulin-like PPIases (e.g., Ess1 and Pin1).

Pin1 (protein interacting with NIMA (never in mitosis A)-1) specificallyisomerizes phosphorylated Ser/Thr-Pro (pSer/Thr-Pro) motifs of certainpolypeptides, which is important because proline-directed kinases (e.g.,protein kinases that phosphorylate certain Ser/Thr residues that precedea proline residue) and phosphatases are conformation-specific andgenerally act only on the trans conformation. Pin1 has a two-domainstructure that includes an N-terminal WW domain and a C-terminal PPIasedomain, and structure-function analyses have shown that the uniquesubstrate specificity of Pin1 towards specific pSer/Thr-Pro motifsresults from interactions provided by both the WW domain and the PPIasedomain. The PPIase activity of Pin1 facilitates the regulation of, forexample, growth-signal responses, cell-cycle progression, cellularstress responses, neuronal function, and immune responses.

Exemplary substrates of Pin1, each containing motifs capable of beingisomerized, are listed in Table 1. The functional consequences ofisomerization of the substrates are also listed.

TABLE 1 Pin1 Substrates Functional Consequence Substrate (GenBank ofPPIase Activity Accession Number) Targeting Site(s) of Pin1 UponSubstrate G2/M and Mitotic Regulation NIMA (P11837) — Regulation ofmitotic function RAB4 (NP_004569) — — CDC25 (AAA58417) pThr48/67-ProDephosphorylation and regulation of activity WEE1 (NP_003381) pT186-PInhibition of WEE1 activity PLK1 (P53350) — — MYT1 (NP_004194) — — CDC27(AAH11656) — — CENP-F (P49454) — — Incenp (NP_064623) — — RPB1(CAA65619) pSer5-Pro Regulation of CTD dephosphorylation NHERF-1(AAA80218) pSer279/301-P Dephosphorylation KRMP1 (NP_057279) pT-1604-PRegulation of mitotic function CK2 (NP_808227) Multiple pSer/ Inhibitionof kinase Thr-Pro sites activity TopoIIα (NP_001058) — Inhibition orinduction of phosphorylation DAB2 (NP_001334) — Dephosphorylation p54nrb(CAA72157) Multiple pSer/ — Thr-Pro sites Sil (CAC14001) Multiple pSer/Regulation of function Thr-Pro sites EMI1 (NP_036309) pS10-PStabilization G1/S Regulation Cyclin D1 (NP_444284) pT286-PStabilization and nuclear localization Ki67 pT234-P — c-Myc (CAA46984)pT58-P Dephosphorylation and destabilization Cyclin E (P24864) pS384-PDestabilization Growth and Oncogenic Signaling c-Jun (AAH06175)pS63/73-P Transactivation B-catenin (P35222) pS246-P Stabilization,protein interaction, and transactivation Cf-2 (NP_034298) —Destabilization NF-κB (AAH33210) pT254-P Stabilization, proteininteraction, and transactivation RAF1 (AAA60247) Multiple pSer/Dephosphorylation Thr-Pro sites and prolonging activation c-Fos(CAA24756) Multiple pSer/ Transactivation Thr-Pro sites RARα(NP_001019980) pS77-P Stabilization and transactivation AIB1/SRC-3 —Transactivation and destabilization HBx (NP_110380) pS41-P Stabilizationand potentiation STAT3 (NP_998827) pS727-P Transactivation DNA Damage,Oxidative Stress Response, and Apoptosis p53 (BAC16799) Multiple pSer/Stabilization and Thr-Pro sites transactivation Bcl-2 (NP_000648)pS70/87-P — p73 (CAA72221) Multiple pSer/ Stabilization and Thr-Prosites transactivation BimEL (AAC39593) pS65-P Stabilization p66^(Shc)(AAH14158) — Mitochondrial import CHE1 (P06276) — DestabilizationNeuronal Survival and Degeneration Tau (NP_058519) pT231-PDephosphorylation pT212-P and protein interaction APP (P05067) pT668-PPromotes non-amyloido- genic APP processing and reduces Aβ productionAPP fragment pT668-P Increases Aβ production from C99 APP fragmentSynphilin-1 (AAD30362) pS211/215-P Protein interaction Gephyrin(CAC81240) pS188/194/200-P Protein interaction MCL1 (CAI15504) pT163-PStabilization Immune Response and Asthma NFAT (NP_666017) — AUF1(NP_112738) — Protein interaction IRF3 (AAH71721) pS339-PDestabilization BTK (CAI42359) pS21/115-P Destabilization OthersSIN2-RPD3 — Reduces histone deacetylases hSpt5 (NP_001124297) —

The importance of phosphorylation-independent prolyl isomerization hasalso been documented. For example, the PPIase CypA catalyzes thecis-trans isomerization of the prolyl bond at position Gly237-Pro238 ofthe Crk protein. Other PPIase substrates isomerized in aphosphorylation-independent manner include, without limitation, steroidreceptors, c-Myb, H3P30, H3P38, Itk, 5-hydroxytryptamine type 3 (5-HT3)receptors, the phage tip protein G3P, the Gag polyprotein of the humanimmunodeficiency virus-1 (HIV-1) virion, intracellular calcium releasechannel, CrkII/CrkL proteins, centrosome protein 55 kDa (Cep55), theretroviral Re1 proteins, PKB/Akt, human T-cell leukemia virus type 1(HTLV-1) Tax oncoprotein, Stat3, HER2/Neu, Notch, FAK, FOXO, PML, C/EBP,and SMRT. Deregulation of PPIase activity (e.g., the upregulation ordownregulation of PPIase activity (e.g., an increase or decrease inPPIase activity)) may, for example, result in a greater cis or transcontent of Ser/Thr-Pro motifs present in PPIase substrates, which mayaffect the function of the PPIase substrate and result in thedevelopment of, e.g., cellular proliferation disorders, neurologicaldisorders, asthma, or aging-associated disorders.

Conformation-Specific Antibodies

The present invention describes methods and compositions for thegeneration and use of conformation-specific antibodies or fragmentsthereof. Conformation-specific antibodies may, for example, specificallybind to the cis or trans conformation of a polypeptide. In a specificembodiment, the conformation-specific antibody of the invention may bindto the cis conformation of a phosphorylated or nonphosphorylated Xaa-Promotif of a polypeptide. The conformation-specific antibody may,alternatively, bind to the trans conformation of a phosphorylated ornonphosphorylated Xaa-Pro motif of a polypeptide. The Xaa-Pro motif maybe a phosphorylated Ser/Thr-Pro motif of a polypeptide (e.g., a Pin1substrate). The binding of a conformation-specific antibody to itsantigen (e.g., a Pin1 substrate) may be useful in the treatment,diagnosis, or monitoring of a disorder or the progression of a disorder.

Methods for the preparation and use of antibodies for therapeuticpurposes are described herein and, for example, in U.S. Pat. Nos.6,054,297; 5,821,337; 6,365,157; and 6,165,464, hereby incorporated byreference.

Antigens

Conformation-specific antibodies of the present invention may begenerated using immunogenic antigens (e.g., antigenic peptides)containing, for example, a phosphorylated or nonphosphorylated Xaa-Promotif, where Xaa is any amino acid residue (e.g., serine or threonine)fixed in a particular conformation (e.g., the cis or trans conformation)or in mixed cis and trans conformations or any other motif or amino acidsequence that is capable of cis/trans isomerization. For example, thecis or trans content of phosphorylated or nonphosphorylatedSer/Thr-Pro-containing antigenic peptides of the invention may be fixedby stereoselective synthesis of (Z)- and (E)-alkene mimics byStill-Wittig and Ireland-Claisen rearrangements (J. Org. Chem., 68:2343-2349, 2003; hereby incorporated by reference). Alternatively, thecis or trans content of phosphorylated or nonphosphorylatedSer/Thr-Pro-containing antigenic peptides of the invention may beincreased or fixed by substituting a proline amino acid residue with aproline analog. Proline analogs include, without limitation,homoproline, pipecolic acid (Pip), dimethyl proline (DMP),azetidine-2-carboxylic acid (Aze), tert-butyl-L-proline (TBP),trans-4-fluoro-L-proline (t-4F-Pro), and cis-4-fluoro-L-proline(c-4F-Pro). The cis or trans content of a given antigen may be analyzedby, for example, nuclear magnetic resonance (NMR) analysis.

Antigenic peptides of the invention may contain a phosphorylated ornonphosphorylated Xaa-Pro motif, wherein Xaa is any amino acid residue(e.g., serine or threonine), which is capable of cis/transisomerization. The antigenic peptide may contain the amino acid residuesof the Xaa-Pro motif of a Pin1 substrate (examples of which are providedin Table 1), with the proline residue substituted for a proline analog.The antigenic peptide may also contain the amino acid residues of theXaa-Pro motif of a full-length polypeptide, wherein the full-lengthpolypeptide is any of the following polypeptides or any isoform thereof:steroid receptors, c-Myb (GenBank Accession No. AAA52032), Itk (GenBankAccession No. BAA02873), 5-hydroxytryptamine type 3 (5-HT3) receptors(Gen Bank Accession Nos. NP_001157118, NP_570126, and NP_872395), thephage tip protein G3P, the Gag polyprotein of the human immunodeficiencyvirus-1 (HIV-1) virion (GenBank Accession No. AAD39400), intracellularcalcium release channel, CrkII/CrkL proteins (GenBank Accession Nos.NP_058431, NP_005197, CAG30309, and CAA42199), centrosome protein 55 kDa(Cep55) (GenBank Accession Nos. NP_001120654 and NP_060601), theretroviral Re1 proteins (GenBank Accession No. NP_002899 and ABC40747),PKB/Akt (GenBank Accession No. NP_001014432 and NP_005154), human T-cellleukemia virus type 1 (HTLV-1) Tax oncoprotein (GenBank Accession No.P03409), Stat3 (GenBank Accession No. AAK17196), HER2/Neu (GenBankAccession No. AAD14920), Notch (GenBank Accession Nos. NP_476859), FAK(GenBank Accession Nos. AAA58469, NP_005598, and NP_722560), FOXO(GenBank Accession No. O16850), PML (GenBank Accession No. AAB19601),C/EBP (GenBank Accession Nos. AAA28415 and AAB33475), and SMRT (GenBankAccession Nos. Q9WU42 and AAC50236). The antigenic peptide may furtherinclude additional residues surrounding the Xaa-Pro motif of thefull-length polypeptide. For example, the antigenic peptide may includethe 3-10 amino acid residues N-terminal to the Xaa residue of afull-length polypeptide and the 3-10 amino acid residues C-terminal tothe proline of a full-length polypeptide.

The antigenic peptide of the invention may be, for example, at least 4,5, 6, 7, or 8 amino acid residues in length. The antigenic peptide maybe between 8 and 20 amino acid residues in length (e.g., 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids residues in length) ormay be over 20 amino acid residues in length.

Such antigens may be produced and purified by any of a variety ofmethods known to one of skill in the art. Antigenic peptides may beproduced and purified by, e.g., solid-phase chemical synthesis, in vitrotranscription/translation, or by recombinant technology. The antigenicpeptides may optionally be chemically coupled to a carrier protein orthe peptides may be generated as fusion proteins to increaseantigenicity. Antigenic peptides may be screened based upon theirability to induce the production of conformation-specific antibodies. Inthis respect, such screening techniques may include, but are not limitedto, enzyme-linked immunosorbant assays (ELISA), immunoprecipitation, orother immunoassays.

Exemplary antigens useful in the production of conformation-specificantibodies include antigens containing a phosphorylated ornonphosphorylated Ser/Thr-homoproline, Ser/Thr-Pip, Ser/Thr-DMP,Ser/Thr-Aze, Ser/Thr-TBP, Ser/Thr-t-4F-Pro, Ser/Thr-c-4F-Pro motif.Specific examples of such antigens include, e.g., pThr668-Pip andpThr668-DMP APP peptide (VDAAV-pThr668-Pro-EERHLSK), pThr231-Pip taupeptide, and pThr231-DMP tau peptide (KVAVVR-pThr231-Pro-PKSPS). Otherexemplary antigens are also described in U.S. Patent ApplicationPublication No. 2008/0058276, hereby incorporated by reference. Suchpeptides may be used as antigens for generating, e.g., polyclonal ormonoclonal antibodies (e.g., rabbit or mouse monoclonal antibodies).

Generation and Purification of Conformation-Specific Antibodies

The antigens of the present invention may be used to generate, forexample, monoclonal, polyclonal, chimeric, humanized, or recombinantconformation-specific antibodies by any method known in the art. Thesemethods include the immunological methods described by Kohler andMilstein (Nature 256: 495-497, 1975 and Eur. J. Immunol. 6: 511-519,1976) and Campbell (”Monoclonal Antibody Technology, The Production andCharacterization of Rodent and Human Hybridomas,” in Burdon et al.,Eds., Laboratory Techniques in Biochemistry and Molecular Biology,Volume 13, Elsevier Science Publishers, Amsterdam, 1985), as well as bythe recombinant DNA method described by Huse et al. (Science 246:1275-1281, 1989).

Briefly, the antigens of the present invention may, in combination withan adjuvant, be administered to a host animal (e.g., a rabbit, mouse,goat, sheep, or chicken). The administration of such antigens may beaccomplished by any of a variety of methods, including, but not limitedto, subcutaneous or intramuscular injection. Once administered, theresults of antibody titers produced in the host animal are monitored,which may be conducted by any of a variety of techniques well-known inthe art (e.g., routine bleeds), with the antisera being isolated (e.g.,via centrifugation) and thereafter screened for the presence ofantibodies having a binding affinity for, e.g., the cis or transconformation of a polypeptide or polypeptide fragment. Screening for thedesired antibody may be accomplished by techniques including, e.g.,radioimmunoassays, ELISA, sandwich immunoassays, immunoradiometricassays, gel diffusion precipitation reactions, in situ immunoassays(e.g., using colloidal gold, enzymatic, or radioisotope labels), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays or hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays.

The resultant antisera derived from the host animal may be affinitypurified to derive the antibodies for the present invention. Theantisera may be purified via conventional techniques, such as theintroduction of the antisera onto a separation column. The antigens ofthe present invention may be immobilized on the column to isolate andpurify conformation-specific antibodies. For example, an antigenicpeptide containing a Ser/Thr-DMP motif that is used to generate acis-specific antibody may be immobilized on a column and used to purifythe resulting cis-specific antibody from, e.g., antibodies in the transconformation. The column may then be washed to remove antibodies nothaving specificity for the antigen immobilized on the column, with theremaining conformation-specific antibody ultimately being eluted fromthe column. The isolated conformation-specific antibody may then bestored per conventional practices known to those skilled in the art.

Alternatively, antibody libraries (e.g., naive antibody libraries,synthetic antibody libraries, semi-synthetic antibody libraries, orcombinatorial libraries) may be screened for the identification ofconformation-specific antibodies. Such libraries are commerciallyavailable from a number of sources (e.g., Cambridge Antibody, Cambridge,United Kingdom, Genetastix Corporation, Pacific Northwest Laboratory,Richland, Washington, and MorphoSys AG, Munich, Germany (e.g., HuCalGOLD)). See, e.g., U.S. Pat. Nos. 6,696,248; 6,706,484; 6,828,422; and7,264,963, hereby incorporated by reference.

Screening of an antibody library may be performed by using one of themethods known to one of skill in the art including, e.g., phage-display,selectively infective phage, polysome technology, and assay systems forenzymatic activity or protein stability. Antibodies having the desiredproperty can be identified, for example, by sequencing of thecorresponding nucleic acid sequence, by amino acid sequencing, or bymass spectrometry. Optimization is performed by replacing sub-sequenceswith different sequences (e.g., random sequences) and then repeating thescreening step one or more times. The antibodies may be screened for,e.g., optimized affinity or specificity for a target molecule (e.g., thecis or trans conformation of a target molecule), optimized expressionyields, optimized stability, or optimized solubility.

Conformation-specific antibodies of the present invention recognize andspecifically bind to, for example, a particular conformation (e.g., thecis or trans conformation) of its complementary antigen. For example, asdescribed herein, the conformation-specific antibody may specificallybind to the cis conformation of a phosphorylated or nonphosphorylatedXaa-Pro motif of a polypeptide (e.g., a Ser/Thr-Pro motif of a Pin1substrate), but will not specifically bind to the trans conformation ofthe phosphorylated or nonphosphorylated Xaa-Pro motif of thepolypeptide. In this case, the Ka between the conformation-specificantibody and its antigen is, for example, at least about 10⁻⁴ M, 10⁻⁵ M,10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or greater.In addition to the binding specificity, the conformation-specificantibody will have, for example, at least 10- to 100-fold greateraffinity to one conformation (e.g., the cis conformation) than toanother conformation (e.g., the trans conformation) of the Xaa-Promotif. The conformation-specific antibody may have, for example, atleast 10³-, 10⁴-, 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹-, or 10¹⁰-fold greateraffinity to one conformation (e.g., the cis conformation) than anotherconformation (e.g., the trans conformation).

Therapeutic Formulations

The conformation-specific antibodies of the present invention may beused in the treatment, inhibition, or prevention of disorders associatedwith the deregulation of PPIase (e.g., Pin1) activity. Theconformation-specific antibodies may also be used to ameliorate symptomsof these disorders. Such disorders include, for example, cellularproliferation disorders (e.g., cancer), neurological disorders (e.g.,Alzheimer's disease), aging-related disorders, asthma, microbialinfections (e.g., viral infections (e.g., HIV infections)), and aging orother aging-related disorders.

The conformation-specific antibodies of the present invention can beformulated and administered in a variety of ways (e.g., routes known forspecific indications, including, but not limited to, topically, orally,subcutaneously, bronchial injection, intravenously, intracerebrally,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, intraarterially, intralesionally,parenterally, intraventricularly in the brain, or intraocularly). Forexample, the pharmaceutical composition containing theconformation-specific antibody may be in the form of a pill, tablet,capsule, liquid, or sustained-release tablet for oral administration; aliquid for intravenous or subcutaneous administration; a polymer orother sustained-release vehicle for local administration; or anointment, cream, gel, liquid, or patch for topical administration.

Continuous systemic infusion or periodic injection of theconformation-specific antibody can be used to treat or prevent adisorder. Treatment can be continued for a period of time ranging fromone day through the lifetime of the subject, for example, 1 to 100 days,1 to 60 days, or until the symptoms of the disorder are reduced orremoved. Dosages vary depending on the severity of the disorder orsymptoms of the disorder. Sustained-release systems and semipermeable,implantable membrane devices are also useful as a means for deliveringthe pharmaceutical composition of the invention. In another embodiment,the composition is administered locally, e.g., by inhalation, and thisadministration can be repeated periodically.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueous solutions(see, e.g., Remington's Pharmaceutical Sciences, 20^(th) edition, Ed. A.Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).Acceptable carriers include, e.g., saline; buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagines, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably atphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments, the preservative concentration ranges from 0.1 to 2.0%v/v. Suitable preservatives include those known in the pharmaceuticalarts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparabenare preferred preservatives. Optionally, the formulations of theinvention can include a pharmaceutically acceptable surfactant.Preferred surfactants are non-ionic detergents. Preferred surfactantsinclude Tween-20 and pluronic acid (F68). Suitable surfactantconcentrations are, e.g., 0.005 to 0.02%.

The conformation-specific antibodies of the invention are administeredto the subject in therapeutically effective amounts. Preferably, theantibodies are administered parenterally or intravenously by continuousinfusion. The dose and dosage regimen depends upon the severity of thedisorder and the overall health of the subject. The amount of antibodyadministered is typically in the range of about 0.001 to about 10 mg/kgof subject weight, preferably 0.01 to about 5 mg/kg of subject weight.

For parenteral administration, the conformation-specific antibodies areformulated in a unit dosage injectable form (e.g., solution, suspension,or emulsion) in association with a pharmaceutically acceptableparenteral vehicle. Such vehicles are inherently non-toxic andnon-therapeutic. Examples of such vehicles include, e.g., water, saline,Ringer's solution, dextrose solution, and 5% human serum albumin.Nonaqueous vehicles, such as fixed oils and ethyl oleate, may also beused. Liposomes may be used as carriers. The vehicle may contain minoramounts of additives, such as substances that enhance isotonicity andchemical stability (e.g., buffers and preservatives). The antibodiestypically are formulated in such vehicles at concentrations of about 1mg/ml to 10 mg/ml.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thesubject's disorder; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the subject'sphysician. Wide variations in the needed dosage are to be expected inview of the variety of polypeptides and fragments available and thediffering efficiencies of various routes of administration. For example,oral administration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art. Administrations can besingle or multiple (e.g., 2-, 3-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, ormore administrations). The composition can be administered at anytime(e.g., after diagnosis or detection of a disorder or a conditionassociated with the disorder (e.g., using the diagnostic methods knownin the art or described herein) or before diagnosis of a disorder to asubject at risk of developing the disorder). Encapsulation of theantibody in a suitable delivery vehicle (e.g., polymeric microparticlesor implantable devices) may increase the efficiency of delivery,particularly for oral delivery.

Where sustained release administration of the conformation-specificantibody is desired in a formulation with release characteristicssuitable for the treatment of any disorder requiring administration ofthe antibody, microencapsulation of the antibody may be contemplated.Microencapsulation of polypeptides for sustained release has beensuccessfully performed with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120 (see, e.g., Johnson et al., Nat.Med. 2: 795-799, 1996; Yasuda, Biomed. Ther. 27: 1221-1223, 1993; Horaet al., Bio/Technology 8: 755-758 1990; Cleland, “Design and Productionof Single Immunization Vaccines Using Polylactide PolyglycolideMicrosphere Systems,” in “Vaccine Design: The Subunit and AdjuvantApproach,” Powell and Newman, Eds., Plenum Press: New York, pp. 439-462,1995; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No.5,654,010, hereby incorporated by reference).

The sustained-release formulations may include those developed usingpoly-lactic-coglycolic acid (PLGA) polymer. The degradation products ofPLGA, lactic and glycolic acids, can be cleared quickly from the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition (see,e.g., Lewis, “Controlled release of bioactive agents fromlactide/glycolide polymer,” in M. Chasin and Dr. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, pp. 1-41, 1990).

The antibody for use in the present invention may also be modified in away to form a chimeric molecule comprising a conformation-specificantibody fused to another heterologous polypeptide or amino acidsequence, such as an Fc sequence or an additional therapeutic molecule(e.g., a chemotherapeutic agent).

The conformation-specific antibody of the present invention may bepackaged alone or in combination with other therapeutic compounds as akit. Non-limiting examples include, e.g., kits that contain, e.g., onepill, two pills, a powder (optionally in combination with a pill ortablet), a suppository and a liquid in a vial, or two topical creams.The kit can include optional components that aid in the administrationof the unit dose to patients, such as vials for reconstituting powderforms, syringes for injection, customized IV delivery systems, orinhalers. Additionally, the unit dose kit can contain instructions forpreparation and administration of the compositions. The kit may bemanufactured as a single-use unit dose for one subject, multiple dosesfor a particular subject (e.g., at a constant dose or in which theindividual compounds may vary in potency as therapy progresses), or thekit may contain multiple doses suitable for administration to multiplesubjects (e.g., “bulk packaging”). The kit components may be assembledin cartons, blister packs, bottles, tubes, or vials.

Combination Therapies

The conformation-specific antibodies may be provided in conjunction(e.g., before, during, or after) with additional therapies to treat adisorder (e.g., a cellular proliferation disorder, a neurologicaldisorder, asthma, or a microbial infection). Treatment therapies thatcan be used in combination with the methods of the invention include,but are not limited to, chemotherapeutic agents, anti-inflammatoryagents, antimicrobial agents, analgesics and anesthetics,bronchodilators, agents for the treatment of neurological disorders, andPPIase inhibitors.

Chemotherapeutic Agents

Any suitable chemotherapeutic agent may be administered in combinationwith the conformation-specific antibody. Chemotherapeutic agentssuitable for the composition described herein include, e.g.,asparaginase, bleomycin, busulfan carmustine (BCNU), chlorambucil,cladribine (2-CdA), CPT11, cyclophosphamide, cytarabine (Ara-C),dacarbazine, daunorubicin, dexamethasone, doxorubicin (adriamycin),etoposide, fludarabine, 5-fluorouracil (5FU), hydroxyurea, idarubicin,ifosfamide, interferon-α (native or recombinant), levamisole, lomustine(CCNU), mechlorethamine (nitrogen mustard), melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, paclitaxel, pentostatin,prednisone, procarbazine, tamoxifen, taxol-related compounds,6-thioguanine, topotecan, vinblastine, and vincristine. Exemplarychemotherapeutic agents are listed in, e.g., U.S. Pat. Nos. 6,864,275and 6,984,654, hereby incorporated by reference.

Anti-Inflammatory Agents

Any suitable anti-inflammatory agent may be administered. Suitableanti-inflammatory agents include, e.g., non-steroidal anti-inflammatorydrugs (e.g., ibuprofen or tacrolimus), cyclooxygenase-2-specificinhibitors such as rofecoxib (Vioxx®) and celecoxib (Celebrex®), topicalglucocorticoid agents, and specific cytokines directed at T lymphocytefunction. Additional suitable anti-inflammatory agents includeflubiprofen, diclofenac, and ketarolac. Anti-inflammatory concentrationsknown to be effective may be used. For example, ibuprofen may be presentin the composition at concentrations sufficient to deliver between25-800 mg per day to the subject. Exemplary anti-inflammatory agents arelisted in, e.g., U.S. Pat. Nos. 7,112,578 and 7,199,119, herebyincorporated by reference.

Antimicrobial Agents

Any of the many known antimicrobial agents can be used in thecompositions described herein at concentrations generally used for theseagents. Antimicrobial agents include, e.g., antibacterials, antifungals,and antivirals.

Examples of antibacterial agents (e.g., antibiotics) include penicillins(e.g., penicillin G, ampicillin, methicillin, oxacillin, andamoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime,and ceftriaxone), tetracyclines (e.g., doxycycline, minocycline, andtetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin,neomycin, streptomycin, and tobramycin), macrolides (e.g., azithromycin,clarithromycin, and erythromycin), fluoroquinolones (e.g.,ciprofloxacin, lomefloxacin, moxifloxacin, and norfloxacin), and otherantibiotics including chloramphenicol, clindamycin, cycloserine,isoniazid, rifampin, and vancomycin. Exemplary antimicrobial agents arelisted in, e.g., U.S. Pat. Nos. 6,830,745 and 7,056,917, herebyincorporated by reference.

Antiviral agents are substances capable of destroying or suppressing thereplication of viruses. Examples of antiviral agents include1-β-D-ribofuranosyl-1,2,4-triazole-3 carboxamide (ribavirin),9-2-hydroxy-ethoxy methylguanine, adamantanamine,5-iodo-2′-deoxyuridine, trifluorothymidine, interferon, adeninearabinoside, protease inhibitors, thymidine kinase inhibitors, sugar orglycoprotein synthesis inhibitors, structural protein synthesisinhibitors, attachment and adsorption inhibitors, and nucleosideanalogues such as acyclovir, penciclovir, valacyclovir, and ganciclovir.Exemplary antiviral agents are listed in, e.g., U.S. Pat. Nos. 6,093,550and 6,894,033.

Antifungal agents include both fungicidal and fungistatic agents, e.g.,amphotericin B, butylparaben, clindamycin, econaxole, fluconazole,flucytosine, griseofulvin, nystatin, and ketoconazole. Exemplaryantifungal agents are listed in, e.g., U.S. Pat. Nos. 5,627,153 and7,125,842, hereby incorporated by reference.

Analgesics and Anesthetics

Any of the commonly used topical analgesics and anesthetics can be usedas therapeutic agents in the invention. Examples of useful anestheticsinclude procaine, lidocaine, tetracaine, dibucaine, benzocaine,p-buthylaminobenzoic acid 2-(diethylamino) ethyl ester HCl, mepivacaine,piperocaine, and dyclonine. Exemplary anesthetics are listed in, e.g.,U.S. Pat. Nos. 6,562,363 and 6,569,839, hereby incorporated byreference.

Analgesics include opioids such as, e.g., morphine, codeine,hydrocodone, and oxycodone. Any of these analgesics may also beco-formulated with other compounds having analgesic or anti-inflammatoryproperties, such as acetaminophen, aspirin, codeine, naproxen, andibuprofen. Exemplary analgesics are listed in, e.g., U.S. Pat. Nos.6,869,974 and 7,202,259, hereby incorporated by reference.

Bronchodilators

Any commonly used bronchodilator can be used as a therapeutic agent inthe invention described herein. Examples of useful bronchodilatorsinclude, e.g., pirbuterol, epinephrine, albuterol, salbutamol,salmeterol, or levalbuterol. Exemplary bronchodilators are listed in,e.g., U.S. Pat. Nos. 4,489,078, 4,591,588, 4,734,413, 6,299,863, and6,555,583, hereby incorporated by reference.

Agents for the Treatment of Neurological Disorders

Agents for the treatment of neurological disorders may be used incombination with the therapeutic compositions described herein.Exemplary agents used for the treatment of such disorders includehaloperidol, carbamazepine, valproate, donepezil, galanthamine, NMDAantagonists (e.g., memantine), PDE4 inhibitors (e.g., Ariflo),γ-secretase inhibitors, β-secretase inhibitors, GSK-3-α inhibitors,compounds which inhibit the aggregation of Aβ, carbidopa/levodopa,entacapone, tolcapone, pramipexole, ropinerole, pergolide,bromocriptine, selegeline, amantadine, vitamin E, amantadine, coenzymeQ, and anticholingergic agents.

PPIase Inhibitors

PPIase inhibitors include, for example, PiA(2,7-dimethylbenzophenanthroline-1,3,6,8 (2H,7H)-tetrone), PiB(diethyl-1,3,6,8-tetrahydro-1,3,6,8-tetraoxobenzo-phenanthroline-2,7-diacetate),PiJ(diethyl-1,3,8,10-tetrahydro-1,3,8,10-tetraoxo-anthra[2,1,9-def:6,5,10-d′e′f′]diisoquinoline-2,9-diacetate),cyclosporin A, FK506, ascomycin, and rapamycin. Additional PPIaseinhibitors are described in U.S. Pat. No. 6,462,173 and U.S. PatentApplication Publication No. 2004/0171019, hereby incorporated byreference.

Diagnostics

The present invention features methods and compositions to treat,diagnose, and monitor the progression of a disorder described herein(e.g., a cellular proliferation disorder, a neurological disorder, anaging-related disorder, asthma, or a microbial infection). The methodsand compositions can include the detection and measurement of, forexample, Pin1 substrates (or any fragments or derivatives thereof)containing a phosphorylated Ser/Thr-Pro motif in a cis or transconformation. The methods can include measurement of absolute levels ofthe Pin1 substrate in a cis or trans conformation as compared to anormal reference. For example, a serum level of a Pin1 substrate in thecis or trans conformation that is less than 5 ng/ml, 4 ng/ml, 3 ng/ml, 2ng/ml, or less than 1 ng/ml serum is considered to be predictive of agood outcome in a patient diagnosed with a disorder (e.g., a disorderassociated with a deregulation of Pin1 activity). A serum level of thesubstrate in the cis or trans conformation that is greater than 5 ng/ml,10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, or 50 ng/ml is considereddiagnostic of a poor outcome in a subject already diagnosed with adisorder, e.g., associated with a deregulation of Pin1 activity.

For diagnoses based on relative levels of substrate in a particularconformation (e.g., a Pin1 substrate in the cis or trans conformation),a subject with a disorder (e.g., a disorder associated with aderegulation of PPIase activity) will show an alteration (e.g., anincrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) in theamount of the substrate in, for example, the cis conformation. A normalreference sample can be, for example, a prior sample taken from the samesubject prior to the development of the disorder or of symptomssuggestive of the disorder, a sample from a subject not having thedisorder, a sample from a subject not having symptoms of the disorder,or a sample of a purified reference polypeptide in a given conformationat a known normal concentration (i.e., not indicative of the disorder).

Standard methods may be used to measure levels of the substrate in anybodily fluid, including, but not limited to, urine, blood, serum,plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methodsinclude immunoassay, ELISA, Western blotting, and quantitative enzymeimmunoassay techniques.

For diagnostic purposes, the conformation-specific antibodies may belabeled. Labeling of the antibody is intended to encompass directlabeling of the antibody by coupling (e.g., physically linking) adetectable substance to the antibody, as well as indirect labeling theantibody by reacting the antibody with another reagent that is directlylabeled. For example, the antibody can be labeled with a radioactive orfluorescent marker whose presence and location in a subject can bedetected by standard imaging techniques.

The diagnostic methods described herein can be used individually or incombination with any other diagnostic method described herein for a moreaccurate diagnosis of the presence or severity of a disorder (e.g., acellular proliferation disorder or a neurological disorder). Examples ofadditional methods for diagnosing such disorders include, e.g.,examining a subject's health history, immunohistochemical staining oftissues, computed tomography (CT) scans, or culture growths.

Subject Monitoring

The diagnostic methods described herein can also be used to monitor theprogression of a disorder (e.g., a cellular proliferation disorder or aneurological disorder) during therapy or to determine the dosages oftherapeutic compounds. In one embodiment, the levels of, for example,polypeptides (e.g., Pin1 substrates) with pSer/Thr-Pro motifs in the cisor trans conformation are measured repeatedly as a method of diagnosingthe disorder and monitoring the treatment or management of the disorder.In order to monitor the progression of the disorder in a subject,subject samples can be obtained at several time points and may then becompared. For example, the diagnostic methods can be used to monitorsubjects during chemotherapy. In this example, serum samples from asubject can be obtained before treatment with a chemotherapeutic agent,again during treatment with a chemotherapeutic agent, and again aftertreatment with a chemotherapeutic agent. In this example, the level ofPin1 substrate with a pSer/Thr-Pro motif in the cis conformation in asubject is closely monitored using the conformation-specific antibodiesof the invention and, if the level of Pin1 substrate with a pSer/Thr-Promotif in the cis conformation begins to increase during therapy, thetherapeutic regimen for treatment of the disorder can be modified asdetermined by the clinician (e.g., the dosage of the therapy may bechanged or a different therapeutic may be administered). The monitoringmethods of the invention may also be used, for example, in assessing theefficacy of a particular drug or therapy in a subject, determiningdosages, or in assessing progression, status, or stage of the infection.

EXAMPLES Example 1 Synthesis of Conformation-Specific Antibodies

We describe the synthesis and purification of conformation-specificantibodies recognizing cis- or trans-pT231-P tau.

Since about 90% of pSer/Thr-Pro motifs in a synthetic peptide are intrans (FIG. 3A), a major challenge is to increase the cis content in theantigen. We have identified a non-natural amino acid that has a similarstructure to proline, homoproline (PIP). PIP dramatically increases thecis content of the synthetic peptide to about 50% (FIGS. 3A and 3 B).Peptides containing PIP or similar proline analogs (e.g., dmP) aresynthesized according to standard techniques. These peptides arefragments of full-length phosphorylated proteins (e.g., Pin1 substrates)containing Xaa-Pro motifs (e.g., tau protein or APP) with the proline ofthe Xaa-Pro motif of the full-length polypeptide replaced by a prolineanalog in the synthetic peptide. These peptides are used to immunizehost animals (e.g., rabbits). See, e.g., FIG. 3C.

To separate cis- and trans-specific antibodies generated by theimmunized animals, we synthesized a biotinatedpT231-(L-5,5-dimethylproline) (dmP) tau peptide and conjugated it to anaffinity column and purify the cis- and trans-specific antibodiesaccording to the scheme in FIG. 3C. Importantly, the cis-specificantibodies, but not trans-specific antibodies, recognize the pT231-dmPtau peptide (FIG. 3D). Thus, the pT231-dmP column bound cis-specificantibodies, while trans-specific antibodies were found in the unboundfraction. The cis-specific antibody was eluted from the column using aThr-Pro peptide. To obtain purified trans-specific antibody, the unboundfraction containing the trans-specific antibody was repurified with apThr-Pro peptide, as trans-specific antibody binds pThr-Pro.

We found that both cis- and trans-specific tau antibodies recognizedpT231-Pro tau peptide with similar intensity (FIG. 3D). Neither cis- nortrans-specific antibodies recognized the nonphosphorylated T231-Pro taupeptide or pT231-Ala tau peptide. Moreover, both cis- and trans-specificantibodies specifically recognized phosphorylated T231-containing tauprotein, but not its Thr231Ala point mutant. Thus, cis- andtrans-specific pT231 tau antibodies have the expected properties andspecificity with little cross-reactivity.

Example 2 Pin1 Overexpression Decreased cis-pT231-tau, but Increasedtrans-pT231-tau in Tau-Tg Mice

We found that the cis/trans ratio of pT231-tau increases duringtauopathy development, but that overexpression of Pin1 can reduce theamount of tau peptide that is in the cis conformation. An AD mouse modeloverexpressing human wild-type tau under the Thy1 promoter (Tau-Tg)develops an age-dependent tauopathy phenotype. We found that only thecis, but not trans, pT231-Tau were dramatically accumulated in agedbrains (FIGS. 4A, C, and D). However, when Tau-Tg mice were crossed withThy1-Pin1 transgenic mice, which reduced endogenous tau levels (FIGS. 4Cand D). Pin1 overexpression not only effectively prevented accumulationof cis pT231-Tau, but also increased trans, pT231-Tau (FIGS. 4B, C, andD), as documented both by immunostaining and immunoblotting analyses.These conformation-specific results provide the first in vivo evidencethat Pin1 promotes cis-to-trans isomerization of pTau to protect againsttangle formation. These results also suggest that it is not generalpT231-Tau, but rather its cis/trans ratio, that is pathologicallysignificant in the tauopathy development in mouse models.

Example 3 Elevation of cis-, but not trans-, pT231-tau in DegeneratedHuman Brains

We found that cis-, but not trans-, pT231-tau is elevated in subjectswith mild cognitive impairment (MCI) and AD.

To examine changes in pT231-tau conformation at different AD stages inhumans, we immunostained normal and AD brain tissue with cis- ortrans-specific tau antibodies. There was little cis- or trans-pT231-taupresent in normal human brains (FIGS. 5A and 5B). In AD, trans-pT231-tauwas barely detectable. Even at Braak VI, very few neurons displayed astrong trans-pT231-tau signal (FIG. 5A). In contrast, cis-pT231-tau wasdetected and found to accumulate in somatodendritic regions of neuronsat Braak III or IV (MCI) brain tissue (FIG. 5B). Furthermore,cis-pT231-tau continued to accumulate as Braak stage increased (FIGS. 5Band 5C). These results showed that cis-pT231-tau is accumulated at anearly stage of degeneration before AD pathologies develop.

To confirm these results, we compared brain tissue immunostained witheither cis-pT231-tau antibodies or mAb TG3. Brain tissue immunostainedwith TG3 showed strong signals only in Braak stage V AD brain tissue,but not in Braak III or IV MCI brain tissue (FIG. 5D), confirming thatTG3 recognizes tau phosphorylated on T231 only in the AD-specificconformation. However, cis-pT231-tau was readily detected in Braak IIIor IV MCI brain tissue (FIG. 5D). These results show that cis-, but nottrans-, pT231-tau is elevated at very early stages of AD and furtheraccumulates as the disease progresses in human brains.

Example 4 The cis/trans Ratio of pT231-tau in the Cerebrospinal Fluid(CSF) of AD Patients

We found that the cis/trans ratio of pT231-tau in the cerebrospinalfluid (CSF) was elevated in late AD patients with small individualvariations.

To examine whether it is possible to assay cis- and/or trans-pT231-tauin CSF, we obtained postmortem CSF from five late AD patients and twocontrol subjects (courtesy of Dr. Neil Kowall) and performed an assayusing the INNOTEST hTau ELISA kit (Innogenetics) to detect the presenceof cis- and trans-pT231-tau. The detection antibodies in the kit werereplaced with cis- or trans-pT231-tau polyclonal antibodies. Althoughneither cis- nor trans-pT231-tau was detectable in control CSF, cis- andtrans-pT231-tau were detected in the CSF of AD patients (p<0.0001) andshowed large individual variations. However, variations in the cis/transratio values of pT231-tau were smaller (from >10-fold to <0.5-fold).These results show that, unlike the pT231-tau peptide (FIG. 3D), thecis- and trans-pT231-tau present in CSF do not have the sameimmunoreactivity to the cis and trans antibodies, indicating thefeasibility of quantifying cis- and trans-pT231-tau proteins in CSF.These preliminary results show that both cis- and trans-pT231-tauproteins are elevated in the CSF of AD patients and suggest thatcis/trans ratio of pT231-tau can serve as a biomarker for AD.

Example 5 Monoclonal Antibodies Recognizing cis- and trans-pT231-tau

To establish cis- and trans-pT231-tau conformations as biomarkers for ADdiagnosis, it is important to produce monoclonal antibodies thatdistinguish cis- and trans-pT231-Pro motif in tau. We immunize rabbitswith pT231-Prx tau peptide (KVAVVR-(pT231)-(Prx)-PKSPS) and screenhybridoma clones producing antibodies recognizing pT231-tau in the cisor trans conformation using various in vitro and in vivo proceduresknown to one of skill in the art.

Example 6 Levels of cis- and trans-pT231-tau in Brain Tissue and CSF atDifferent Stages of AD

Human AD brain tissue samples and normal controls are obtained frombrain autopsy and ventricular CSF samples collected from the patientswith Braak I-VI stage disease. The levels of cis- and trans-pT231-tau inCSF samples are measured using INNOTEST hTau ELISA kit (Innogenetics),replacing the detection antibody with cis- and trans-pT231-taupolyclonal antibodies or mAb. A pT231-tau synthetic peptide is used as astandard. Simultaneous measurement of t-tau, pT231-tau, and Aβ1-42 inCSF has been well established using the multiplex xMAP Luminex platformwith Innogenetics' immunoassay kit-based reagents (INNO-BIA AlzBio3;Ghent, Belgium). Alternatively, it is possible to replace the pT231detection antibody CP9 with cis- and trans-specific antibodies tosimultaneously measure cis- and trans-pT231-tau levels with t-tau andAβ1-42. Calibration curves are produced for each biomarker using aqueousbuffered solutions that contain the combination of three biomarkers atdifferent concentrations of recombinant tau, synthetic Aβ1-42 peptide,and pT231-tau synthetic peptide as standards. Assays are carried out intriplicate for each sample and the OD values imported into SPSSanalytical software and transformed into concentrations according to thestandard curve for statistical analysis.

To understand the relationship between pT231-tau conformations in CSFand in brain tissue, ELISA is used to quantify cis- and trans-pT231-taulevels in brain lysates. Immunostaining and immunoblotting analyses areperformed on frontal cortex tissues from the same individuals whose CSFsamples are analyzed to confirm the ELISA results. To examine therelationship between pT231-tau conformations and other tau-relatedpathologies, the relationship between cis- and trans-pT231-tau and thepresence of the pretangle pathology and/or neurofibrillary lesion isdetermined. The pre-tangle pathology is detected by immunostaining brainsections with various phospho-specific and/or conformation-specific tauantibodies or by extracting tau from brains using sarcosyl, followed byimmunoblotting analysis with various tau antibodies. The presence ofneurofibrillary lesions and neurodegeneration is detected by Gallyassilver staining, thioflavin-S staining, NeuN staining, and Nisslstaining. To compare the changes of tau conformations and theirrelationship with other tauopathy phenotypes at different Braak stages,immunoblotting data is quantified with imagequant or immunofluoresencestaining, using Zeiss LSM510 META imaging system and software forstatistically relevant analysis. A comparative study of cis- andtrans-pT231-tau, pT231-tau, total tau, and Aβ1-42 levels at variousBraak stages, among AD and other different dementia groups, or amongother quantitative measures of AD progression (such as age-at-onset(AAO), disease duration, and Mini-Mental State Examination (MMSE) score)is completed by using Student t test or by one-way analysis of variancefollowed by Bonferroni post hoc test when multiple comparisons areperformed. All data analyses are performed with statistical software.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications, patent applications, and patents mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent application, or patent wasspecifically and individually indicated to be incorporated by reference.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention; can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

What is claimed is: 1-65. (canceled)
 66. A method of treating a subjectwith a disorder, wherein said disorder is associated with a deregulationof PPIase activity, said method comprising administering to said subjecta pharmaceutical composition comprising a conformation-specific antibodyor fragment thereof that specifically binds to the cis conformation of aXaa-Pro motif of a polypeptide present in said subject, wherein said Xaais serine or threonine and is phosphorylated, and said antibody orfragment thereof binds to the cis conformation of said Xaa-Pro motif ofsaid polypeptide with at least 10- to 100-fold reater affinit than tothe trans conformation of said Xaa-Pro motif of said polypeptide, anyand wherein said conformation-specific antibody is administered inamount sufficient to treat said disorder. 67-69. (canceled)
 70. Themethod of claim 66, wherein said PPIase is Pin1.
 71. The method of claim66, wherein said method further comprises administering an additionaltherapeutic agent.
 72. The method of claim 71, wherein said additionaltherapeutic agent is a chemotherapeutic agent.
 73. The method of claim66, wherein said disorder is a cell proliferation disorder or aneurological disorder,
 74. The method of claim 73, wherein said cellproliferation disorder is cancer.
 75. The method of claim 73, whereinsaid disorder is a neurological disorder.
 76. The method of claim 66,wherein said disorder is asthma.
 77. The method of claim 66, whereinsaid disorder is a microbial infection.
 78. The method of claim 66,wherein said disorder is aging or an aging-related disorder. 79-91.(canceled)
 92. The method of claim 71, wherein said additionaltherapeutic agent is an anti-inflammatory agent.
 93. The method of claim71, wherein said additional therapeutic agent is PPIase inhibitor. 94.The method of claim 93, wherein said additional therapeutic agent is anagent for the treatment of neurological disorders.
 95. The method ofclaim 74, wherein said cancer is selected from the group consisting ofprostate cancer, squamous cell cancer, small-cell lung cancer,non-small-cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, gastric cancer, and melanoma.96. The method of claim 75, wherein said neurological disorder isselected from the group consisting of Alzheimer's disease, mildcognitive impairment, Parkinson's disease, multiple sclerosis, musculardystrophy, corticobasal degeneration, dementia pugilistica, Down'ssyndrome, frontotemporal dementias, myotonic dystrophy, Niemann-Pickdisease, Pick's disease, prion disease, progressive supranuclear palsy,subacute sclerosing panencephalistis, epilepsy, vascular dementia,age-related dementia, head trauma, stroke, neurofibromatosis, Lewy bodydisease, amyotrophic lateral sclerosis, peripheral neuropathies, andmacular degeneration.
 97. The method of claim 66, wherein saidpolypeptide is a PPIase substrate.
 98. The method of claim 97, whereinsaid PPIase substrate is a Pin1 substrate.
 99. The method of claim 98,wherein said Pin1 substrate is NIMA, RAB4, CDC25, WEE1, PLK1, MYT1,CDC27, CENP-F, Incenp, RBP1, NHERF-1, KRMP1, CK2, TopoIIα, DAB2, p54nrb,SiI, EMI1, cyclin D1, Ki67, c-Myc, cyclin E, c-Jun, β-catenin, Cf-2,NF-κB, RAF1, c-Fcs, RARα, AIB1/SRC-3, HBx, STAT3, p53, Bcl-2, p73,BimEL, p66Shc, CHE1, tau, amyloid precursor protein (APP), APP fragment,synphilin-1, gephyrin, MCL1, NFAT, AUF1, IRF3, BTK, SIN3-RPD3, or hSpt5.100. The method of claim 66, wherein said antibody is a monoclonalantibody.
 101. The method of claim 66, wherein said antibody is ahumanized antibody.